In living cells, complex processes are typically accomplished by highly specific binding interactions among functional cell components, most commonly involving one or more proteins. Understanding which proteins bind to one another, and under what circumstances, poses difficult unsolved problems. An approach to learning which proteins bind to each other to form protein complexes is to isolate functional protein complexes, or portions thereof, in order to identify their components.
However, the dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. Those low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. In fact, most in vivo protein-protein binding is transient and occurs only briefly to facilitate signaling or metabolic function. Capturing or freezing those momentary contacts to study which proteins are involved and how they interact is difficult.
For example, protein kinases and their substrates represent the largest signaling network that regulates multiple cellular functions. Deregulation of kinase activity has emerged as a major mechanism in numerous diseases. Kinase signaling cascades involve a complicated array of interconnected networks that are reprogrammed in response to disease events and in the presence of inhibitors. Typical, high throughput approaches such as yeast two-hybrid screening or mass spectrometry-based immunoaffinity purification are ineffective for the identification of unknown kinase substrates due to high false-positive rates and inability to detect their transient, dynamic interactions.